Download Beneficial and harmful roles of bacteria from the Clostridium genus*

Vol. 60, No 4/2013
515–521
on-line at: www.actabp.pl
Review
Beneficial and harmful roles of bacteria from the Clostridium
genus*
Dorota Samul*, Paulina Worsztynowicz, Katarzyna Leja and Włodzimierz Grajek
Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, Poznań, Poland
Bacteria of the Clostridium genus are often described
only as a biological threat and a foe of mankind. However, many of them have positive properties and thanks to
them they may be used in many industry branches (e.g.,
in solvents and alcohol production, in medicine, and
also in esthetic cosmetology). During the last 10 years
interest in application of C. botulinum and C. tetani in
medicine significantly increased. Currently, the structure
and biochemical properties of neurotoxins produced by
these bacterial species, as well as possibilities of application of such toxins as botulinum as a therapeutic factor in humans, are being intensely researched. The main
aim of this article is to demonstrate that bacteria from
Clostridium spp. are not only pathogens and the enemy
of humanity but they also have many important beneficial properties which make them usable among many
chemical, medical, and cosmetic applications.
Key words: Clostridium spp., biotechnological applications, medical
and cosmetic importance, pathogenicity
Received: 18 October, 2013; revised: 02 December, 2013; accepted:
05 December, 2013; available on-line: 29 December, 2013
INTRODUCTION
Bacteria of the Clostridium genus are anaerobic,
Gram-positive, rod-shaped, endospore-forming bacteria. They have been known for a very long time. The
first information about them is presented in Epidemics
III —– the book described by Hippocrates in 430–370
BC. Hippocrates reported on C. histoliticum as a cause
of gas gangrene (Mayr, 1969). The main symptom of
this disease is swelling of the skin (Nigel et al., 2001).
Other diseases caused by C. tetani were described by
Charles Bell in his book. The information about other diseases caused by C. tetani appeared in Essays on
the Anatomy and Philosophy of Expression in 1824 (Mayr,
1969). However, bacteria from the Clostridium genera
were recognized as a separate starter culture were observed only in the 19th century by Louis Pasteur. He
was the first to recognize that bacteria can exist and
grow without oxygen. His discovery was a huge revelation in his time. Pasteur called such microorganisms
Vibrion butyrique because of the main product of their
fermentation pathway and introduced the word “anaerobic” to name life without oxygen. V. butyrique was renamed 20 years later as C. butyricum by another scientist
— Adam Prażmowski (Dürre, 2001).
Bacteria from the Clostridium genera are commonly
present in natural environment, e.g. they live in dust,
soil, water, bottom sediments and in human and animal
alimentary canals (Moriishi et al., 1996). They are most-
ly known as pathogenic microorganisms. However, they
also play an important role in many fields of industrial
metabolite production. Bacteria from Clostridium ssp. are
able to ferment divers organic compounds and produce
large amounts of gases (carbon dioxide, hydrogen, methane), organic acids (lactic, acetic, butyric, and fumaric
acids), and also solvents (butanediol and propanediol)
(Buckel et al., 2005). They are also used in medicine and
esthetic cosmetology. In the past 10 years an interest
in application of C. botulinum and C. tetani in medicine
significantly increased. Currently, the structure and biochemical properties of neurotoxins produced by these
bacteria, as well as the possibilities of application of such
botulinum toxins as a therapeutic factors in humans,
are being intensely researched (Jankovic & Hallet, 1994;
Brin, 1997; Bigalke & Shoer, 2000; Schiavo et al., 2000;
Kreydon et al., 2000; Rosetto et al., 2001; Dressler, 2000).
In the literature one can also find information that bacteria from C. sporogenes, C. butyricum, C. perfingens, C. acetobutylicum and C. botulinum species can be employed in
bacteriocin production (Clarke et al., 1975; Barber et al.,
1979; Eklund et al., 1988).
In the bacterial world, in one family there are positive
and negative bacteria for human and animal species. It
is more interesting when the same bacterial strain plays
a double role and is both: harmful and beneficial. This
article is a review of some possibilities of applications
for the bacteria from Clostridium genera in biotechnological processes (in the chemical and energy industry), in
medicine and cosmetic, and about pathogenicity of these
bacteria.
THE USE OF BACTERIA OF THE CLOSTRIDIUM GENUS
IN THE CHEMICAL INDUSTRY
Bacteria of the Clostridium genus are able to employ
intense fermentation metabolism (Jiang et al., 2009; Masset et al., 2010). They are able to produce wide range of
metabolites, including: 1,3-propanediol (Biebl et al., 1998;
Kubiak et al., 2012), acetic acid, butyric acid, formic
acid (Wu & Yang, 2003; Song, 2011), ethanol, butanol,
acetone (Jones & Woods, 1986; Ezeji et al., 2003), carbon dioxide (Khanal et al., 2004), hydrogen and ammonia (Levin et al., 2006; Ren et al., 2007; Skonieczny &
Yargeau, 2009; Oh et al., 2009; Beckers et al., 2010). The
*
e-mail: [email protected]
*Presented at the 3-rd Workshop on Microbiology „MIKROBIOT
2013” in Łódź, Poland.
Abbreviations: ACh, acetylcholine; BoNT, botulinum neurotoxin;
BoNT-A/BTX-A, botulinum toxin type A; BDEPT, bacterial directed
enzyme prodrug therapy; COBALT, combination of bacteriolytic
therapy; LD50, lethal dose; C., Clostridium; 1,3-PD, 1,3-propanediol;
V., Vibrion; S., Salmonella
516
D. Samul and others
2013
Production of organic acids
Organic acids are low-molecular
weight compounds which are found
in all organisms and which are characterized by the possession of one or
more carboxyl groups (Jones, 1998).
Organic acids are among the candidate replacements for in-feed antibiotics. Some of acids (lactic, acetic) have
a long history of use in food preservatives (Skrivanova et al., 2006). Bacteria
of the genus Clostridium (C. butyricum,
C. beijerinckii, C. populeti, C. propionicum,
C. thermobutyricum, C. tyrobutyricum) are
able to synthesize organic acids: succinic acid, acetic acid, butyric acid and
propionic acid (Johns, 1952; Luers et
al., 1997; Zhu et al., 2002; Willims et
al., 2003). Butyric acid is widely used
in the chemical industry (for the synthesis of polymers of butyryl), pharmaceutical, and especially in the food
industry to strengthen butter notes of
flavor in food products (Zigova et al.,
1999; Wu & Yang, 2003; Liu et al.,
2006). Butyric acid is produced mainly by oxidation of butyraldehyde obtained from oxosynthesis of propylene
Figure 1. Metabolic pathways of bacteria from the Clostridium genera
(Wu & Yang, 2003). Propionic acid is
widely used as an antifungal agent in
food, beer and as an intermediate in
metabolic pathway of production of metabolites by the
the synthesis of herbicides, cellulose acetate (propionate
Clostridium genus bacteria is shown in Fig. 1.
plastics), solvents and pharmaceuticals (Kośmider et al.,
2010). In contrast to propionate production in PropionProduction of 1,3-propanediol
ibacterium, which is produced through decarboxylic acid
1,3-propanediol (1,3-PD) is a three-carbon diol pathway, propionate in C. propionicum can be produced
which consists of two hydroxyl groups on the prima- through the acrylate pathway (Tracy et al., 2012). There
ry carbon atoms, with the formula C3H8O2. 1,3-PD is are two major areas of commercial application of acetic
also known as a trimethylene glycol, 1,3-dihydroxy- acid today: food-grade vinegar and chemically synthepropane, propane-1,3-diol. 1,3-PD is an important sized industrial acetic acid. Among the bacteria of the
chemical by-product in polymer production (Igari et Clostridium genus, C. thermoaceticum is able to synthesize
al., 2000; Drożdżyńska et al., 2011). 1,3-PD is one of acetic acid from xylobiose (Nakamura et al., 2011). Biothe oldest known fermentation products. This diol synthetic routes often have product-specific advantages
was identified in 1881 by August Freund, as a product over chemical synthesis, which are important for extendof glycerol fermentation by C. pasterianum. In the past, ing and adding value to products.
1,3-PD was produced only via chemical methods (Igari et al., 2000). Nowadays, an attractive alternative for Production of solvents
chemical synthesis is a microbial conversion of 1,3Solvents are liquids which form a homogeneous sysPD from renewable resources (Nakamura et al., 2003;
Mu et al., 2006). A number of microorganisms can tem (solution) with the substances dissolved in them.
grow on glycerol as the sole carbon and energy source It is well known for almost 100 years that bacteria can
and produce 1,3-PD. A large group of these micro- produce solvents by fermentation. In the 1920s biotechorganisms are bacteria of the Clostridium genus. This nological production of solvents has been replaced by
group includes: C. diolis, C. acetobutylicum, C. perfingens, petrochemical methods. In the 1960s fermentation which
C. butyricum, C. pasteurianum (Biebl et al., 1998; Hao et used bacteria of the Clostridium genus was completely
al., 2008; Kubiak et al., 2012). C. butyricum is the best eliminated from industry. Clostridium ssp. were eliminated
wild strain to produce 1,3-PD (Wilkens et al., 2012). because the process was characterized by low productiviThis strain is characterized by high productivity and ty, low yield and high cost of the product recovery. Nevlittle nutrition requirements (Buckel, 2005; Drożdżyńs- ertheless, this method cannot be forgotten altogether.
Currently, we can see an internationally growing concern
ka et al., 2011).
Bacteria of the Clostridium genus are also able to for the environment and the need to gain independence
synthesize 1,3-PD with the use of metabolic engineer- from the petrochemical industry. This tendency led to
ing. Metabolic engineering of the microbial produc- an increased interest in the production of solvents from
tion of 1,3-PD concerns native producers of 1,3-PD natural raw materials (e.g. corn, soy, molasses, wood hy(C. butyricum) as well as heterologous hosts (C. acetobu- drolysates etc.). A lot of research in this area has made
tylicum) – microorganisms which are able to form 1,3- the process competitive (Jones et al., 1982; Dabrock et
PD via genetic manipulations (Celińska, 2010; Leja et al., 1992; Qureshi et al., 2000).
Bacteria of the Clostridium genus are able to syntheal., 2011).
size solvents such as acetone, butanol and ethanol. This
Vol. 60 Roles of bacteria from the Clostridium genus
group includes: C. acetobutylicum, C. pasteurianum, C. beijerinckii, C. saccharoperbutylacetonicum and C. saccharobutylicum,
C. sporogenes (Johnson et al., 1997; Keis et al., 2001; Bankar et al., 2012; Sun & Liu, 2012; Tracy et al., 2012; Gottumukkala et al., 2013). C. beijerinckii is able to produce
isopropanol in addition to butanol (George et al., 1983;
Chen & Hiu, 1986). Instead, C. pasterianum ferments hydrocarbons to butanol, acetone, carbon dioxide and hydrogen (Heyndrickx et al., 1991; Dabrock et al., 1992).
For the production of solvents, acetylo-CoA and butyrylo-CoA are the key intermediate factors to obtain ethanol and butanol. Acetyl-CoA is the key intermediate factor to produce acetone (Harris et al., 1999; Kasap, 2002).
THE USE OF BACTERIA OF THE CLOSTRIDIUM GENUS
IN THE ENERGY INDUSTRY
Energy is the source of all human activity. In nature,
energy is never lost but it is changing its form. There
are many different ways in which energy can be stored,
converted and amplified for our use (Demirbaş, 2001).
Production of hydrogen
Hydrogen is the element which is a very efficient
source of energy, the energy equal to 33Whg-1. Hydrogen is regarded as one of the most important sources
of energy. It can be also converted into energy in combustion engines, fuel cells and as a component of rocket
fuel. Hydrogen is produced mainly by chemical methods
(Chin et al., 2003). Currently, 90% of total production is
obtained either from methane or electrolysis of water.
However, the ability of using microorganisms in hydrogen production has been intensly investigated. Among
others, direct and indirect biophotolysis, photofermentation and dark fermentation have been examined (Levin,
2006; Lo et al., 2010). Among promising solutions for
biological hydrogen production there is hydrogen fermentation. Among numerous groups of microorganisms
which are able to produce hydrogen, bacteria of the Clostridium genus, especially C. acetobutylicum and C. butyricum,
are typically used (Chin et al., 2003; Chen et al., 2005).
The reaction of molecular hydrogen formation is as
follows (Khanal et al., 2004):
C12H22O11 + 5 H2O 4 CH3COOH + 4 CO2 + 8 H2
C12H22O11 + H2O 2 CH3CH2CH2COOH + 4 CO2 + 4 H2.
However, in this group of microorganisms there are
also strains which are able to produce hydrogen not only
from glycerol , but also from xylose — C. tyrobutyricum
(Liu et al., 2006) and from cellulosic substrates — C.
thermocellum (Levin et al., 2006).
Biomass conversion
Biomass is a term used for all organic material that
stems from plants (McKendry, 2002a). Biomass is the
oldest energy source used by humans. Traditionally, biomass has been utilized through direct combustion and
this process is still widely used in many parts of the
world. Historically, biomass has been dispersed, labor
intensive and land intensive source of energy. Biomass
differs from other alternative energy sources in that
this resource is diversified. Biomass can include wastes,
standing forests and energy crops. It can be converted
to energy through a number of conversion processes
(Demirbaş, 2001). Conversion of biomass to energy can
employ two main technologies — thermochemical and
biological (McKendry, 2002b).
517
Many microorganisms are able to utilize a variety of
carbohydrates for the conversion of lignocellulose biomass to bioenergy. Pectinolytic bacteria of the Clostridium
genus seem to be especially attractive in this aspect of
biomass conversion. In contrast to the hyperthermophilic microorganisms (Caldicellulosiruptor kristjanssonii, Anaerocellum thermophilum), bacteria of the Clostridium genus are
less thermophilic. This group of bacteria includes numerous species that utilize crystalline cellulose, as well
as hemicellulose, as growth substrates (Blumer-Schuette
et al., 2008). C. flavum, C. laniganii during fermentation of
pectins loosen the plant tissue and allow fast separation
of the cellulose fibers (Lanigan, 1959). These groups of
microorganisms are of great importance to biomass degradation.
SOME APPLICATIONS OF THE CLOSTRIDIUM GENUS IN
MEDICINE AND COSMETICS
Over the past few years botulinum neurotoxin
(BoNT) has transformed from a cause of life-threatening
affliction to a medical therapy. In 1978, Dr. Alan Scott
was the first to use BoNT-A in humans for treatment
of strabismus. Nowadays, after elucidating the pharmacological mode of the botulinum toxin action, it has
become possible to use it in a wide spectrum of health
disorders (Mahajan & Brubaker, 2007).
There are seven types of immunologically distinct serotypes of botulinum neurotoxin (BoNT): A, B, C1, D,
E, F, and G. Toxins produced by clostridial bacteria are
high-molecular-weight protein complexes. Each toxin
is antigenically distinct, but they have similar molecular
weights of 150 kD, as well as structures. All the toxins
consist of a light and heavy chain linked by a disulfide
bond. BoNT acts by blocking the release of acetylcholine (ACh) at the neuromuscular junction and it results
in flaccid paralysis of the treated muscle (Johnson &
Bradshaw, 2001; Majid, 2010; Tsui, 1996; Wheelera &
Smith, 2013).
Botulinum toxin is effective in the treatment of
some pain syndromes, e.g. BoNT can selectively weaken painful muscles by interrupting the spasm pain cycle.
BoNT-A is well tolerated in the treatment of chronic
pain disorders in which pharmacotherapy can cause side
effects, such as migraines, chronic lumbar pain, tension
headaches and myofascial pain. The reduction in the
consumption of analgesics and length of action of three
to four months per dose represent other advantages of
its use (Colhado et al., 2009).
Types A and B of the toxins can also be used in the
treatment of strabismus, blepharospas, nystagmus, vocal tics and stuttering, various manifestations of tremor,
facial muscle contraction and many other dystonias, including cervical focal dystonias.
Additionally, injections of the botulinum toxin are
among the latest means of therapy in treatment of neurological diseases (spasticity, in particular cerebral palsy,
Parkinson’s disease, Tourette’s syndrome), gastroenterological diseases (achalasia), urological diseases (detrusorsphincter dyssynergia, detrusor instability, lower urinary
tract dysfunction), ophthalmological (strabismus) or
dermatological diseases (hyperhidrosis, facial flushing).
Treatment of muscle hyperactivity by injecting BTX-A
into selected muscles produces dose-dependent chemical denervation resulting in reduced muscular activity
(Barnes et al., 2011; Friedmana & Potulska, 2001; Graham et al., 2000; Lin, 2007; Sutcliffe et al., 2005; Tsui,
1996). For example, in a recent study of 30 patients with
518
D. Samul and others
Tourette’s syndrome and phonic tics, injection of the
botulinum toxin improved symptoms in 93% and helped
to improve the patients’ quality of life (Sutcliffe et al.,
2005).
Moreover, not only metabolites of bacteria of the
Clostridium genus, but also whole bacterial cells are used
for medical therapy. Several studies have demonstrated
that they can be used as probiotic agents against internal
hemorrhages caused by enterohaemorrhagic Escherichia
coli. Furthermore, they inhibit the growth of C. difficle,
S. typhimurium and Vibrio spp (Nigel et al., 2001; Ranade,
1989).
In recent years, there have been reports on the use of
natural and engineered non-pathogenic bacterial species
as potential antitumor agents, either to deliver tumoricidal molecules or to provide direct tumoricidal effects.
Currently, the interest in alternative methods of cancer
treatment is high because traditional cancer therapies
have limited effectiveness due to poor penetration that
reduces the dose present throughout tumors and due
to the lack of selectivity to cancer cells which results in
causing damage to normal tissue (Minton et al., 1995;
Barbé et al., 2006; St Jean at al., 2008; Patyar at al., 2010).
Bacteria have the potential to overcome these limitations by actively targeting all tumor regions and delivering therapeutic payloads. The high specificity for cells
of solid tumors is based on hypoxic tumors regions.
Normal tissues are well oxygenated which prevents germination and growth of anaerobic bacteria whereas the
hypoxic tumor regions allow the germination of spores
and vegetative cell proliferation. Live, attenuated or engineered non-pathogenic bacterial species are capable
of multiplying selectively in tumors and inhibiting their
growth (Mellaert at al., 2006; Patyar at al., 2010; Wei
at al., 2008). Research on C. novyi (attenuated strain)
showed significant anti-tumor effects, but these experiments also led to death.
The use of combination of bacteriolytic therapy (COBALT) also resulted in anti-tumor effects, but still was
not devoid of animals’ death. C. novyi has been investigated in conjunction with chemotherapy, radiotherapy
and radioimmunotherapy.
Bacteria are also able to produce and secrete proteins,
which can be combined with targeting in order to apply
a focused therapy. Using proteins with different functionality, there are three methods of bacterial treatment
that can be adjusted: enzymatic drug activation, controlled cytotoxicity and biomolecule secretion (St Jean at
al., 2008).
First strategy, called bacterial directed enzyme prodrug
therapy (BDEPT) involves the tumor-specific location
of bacteria to locally activate systemically administered
‘prodrugs’ within the tumor. BDEPT is a two-step therapy. First, recombinant spores carrying the genetic information about recombinant proteins are injected into the
patient’s body and they are targeted to the site of the tumor where the enzyme is expressed. Next, once the levels of enzyme expression are optimal, prodrugs are administered and are converted to a cytotoxic drug by the
expressed enzyme strictly at the tumor site (Lehouritis et
al., 2013). The last strategy involves producing biologically active molecules to induce a physiological response.
Studies have shown that Clostridum can be designed to
produce therapeutically significant levels of a cytokine,
interleukin-2, which causes T-cell-mediated neoplastic
death.
Over the past decade, facial cosmetic procedures have
become more common place in maxillofacial and oral
surgery and dentistry. One of the most often requested
2013
procedures is a treatment with botulinum toxin type A
(BoNT-A), which is known under several names such as
Botox, Dysport.
The most common cosmetic procedure performed
by intramuscular injection of BoNT is aimed to reduce facial wrinkles. Facial wrinkles are formed primarily due to intensive work of leading muscles, wrinkling one’s brows and longitudinal muscle. Treatment
of facial wrinkles involves injecting into the muscle
different doses of the botulinum toxin, which reduces
facial muscle activity. A significant improvement in facial skin tension is observed in approximately 90% of
patients. The first effect of such a therapy is observed
after 1–4 days, and a maximum after 2–3 weeks. The
final effect is obtained after 3–6 months of therapy.
Another procedure carried out with the use of botulinum toxin is correction of Masseteric hypertrophy.
Masseteric hypertrophy usually results from anatomical asymmetry of the jaw, habitual asymmetric use of
the jaw, excessive chewing of gum, clenching during
exercise or sleep, and congenital malformations. The
results of treatment with intramasseter injections of
botulinum toxin have been encouraging and satisfying
to patients (Jaspers et al., 2011; Majid, 2010).
Additionally, the botulinum toxin is used in treatment
of glabellar lines, frontalis muscle, commuter lines, smile
orbital resin and masseter muscle hypertrophy. Recently, the therapeutic utility of the botulinum toxin in the
treatment of excessive sweating was reported (Sutcliffe
et al., 2005).
Pathogenicity
Many bacteria of the Clostridium genus have adverse effects on food products and human health.
The Clostridium genus includes 35 species of pathogens
producing exotoxins, including C. botulinum, C. perfringens, C. tetani, C. barati, C. haemolyticum, C. novyi, C. septicum, C. chauvoei and C. difficile (Moriishi et al., 1996).
The most well-known pathogen of the Clostridium
genus is C. botulinum that causes botulism. Botulism
is caused by a botulinum neurotoxin (BoNT) produced from the anaerobic, spore-forming bacterium
C. botulinum (Shapiro et al., 1998). The most common
cause of poisoning in humans are neurotoxins type
A, B, and E (Bielec & Modrzewska, 2007; Wheeler
& Smith, 2013). BoNT is a very strong poison, for
example it is 1012 more lethal than sodium cyanide.
The estimated LD50, for humans (assuming a body
weight of 70 kg), extrapolated from experiments on
animals, has been estimated to be approximately 70
µg by oral administration, 0.09–0.15 µg to intravenous
administration and 0.7–0.9 µg by inhalation (Majid,
2010; Tsui, 1996). The disease can be divided into six
types — foodborne botulism, wound botulism, infant
botulism, adult intestinal toxemia botulism, inhalation
botulism and iatrogenic botulism (Cherington, 1998;
Bielec & Modrzewska, 2007). All forms of botulism
produce the same distinct clinical syndrome. Botulism
leads to paralysis that usually starts with the muscles
of the face and then spreads towards the limbs. In severe forms, it leads to paralysis of the breathing muscles and causes respiratory failure, which may lead to
death (Aureli et al., 2008; Sobel, 2005).
Foodborne botulism is due intoxication caused by
consuming food contaminated with the botulinum
toxin. However, wound botulism is caused by contamination of wound with C. botulinum spores and subsequent germination of these spores and production of
Vol. 60 Roles of bacteria from the Clostridium genus
toxin in the anaerobic milieu of an abscess. The toxin
is partially absorbed into the blood (Mahajan & Brubaker, 2007; Sobel, 2005).
Infant botulism occurs in infants between 1 week
and 11 months of age and is a combination of infection and intoxication, including ingestion of C. botulinum spores, germination within the gastrointestinal
tract and in vivo production of toxin (Caya et al., 2004;
Jagoda & Renner, 1990). In part due to the immaturity of the child’s intestinal flora and in part due to a
relatively low production of clostridial-inhibiting bile
acid as compared to the adult gastrointestinal tract,
this factor promotes development of the disease. A
significant risk factor for the development of infant
botulism is honey consumption, because 15% to 25%
of honey products harbor botulinum spores. Therefore, children under one year of age should not eat
honey at all (Caya et al., 2004; Moriishi et al., 1996;
Bielec & Modrzewska, 2007).
Adult intestinal toxemia botulism is caused by the colonization of the colon by C. botulinum producing BoNT
in situ and its absorption into the blood. The emergence
of this form of botulism is caused by the presence of
earlier lesions in the gastrointestinal tract: disturbance
of bacterial flora composition after antibiotic therapy, a
condition after a surgical operation or chronic gastrointestinal inflammatory bowel diseases (Bielec & Modrzewska, 2007).
Iatrogenic botulism is an extremely rare form of the
disease that is caused by intake of BoNT A in the form
of injection. The symptoms are usually limited to generalized muscle weakness, slight drooping of eyelids, double
vision or dryness of mucous membranes in the mouth.
Inhalation of botulinum toxin does not occur naturally
in the natural environment. We all know the case from
1962, where in one of the laboratories in Germany three
employees were infected. All those who became ill were
exposed to an aerosol containing BoNT A. All of them
had symptoms similar to foodborne botulism (Bielec &
Modrzewska, 2007).
Like C. botulinum, bacteria of the C. tetani and C.
sordellii species are dangerous for human health. Infections of those bacteria cause acute diseases and pose
difficult clinical challenges. C. tetani causes tetanus.
C. tetani usually enters the body through a wound
and spores start to germinate. Toxins are produced
and disseminated via blood and lymphatics. Tetanus
is characterized by generalized rigidity and convulsive
spasms of skeletal muscles.
C. sordellii infections are usually fatal. Most commonly, these infections occur after trauma, childbirth,
and routine gynecological procedures, but they have
recently been associated with medically induced abortions and injection drug use (Aldape et al., 2006).
C. difficle causes nosocomial infections associated
with food. Strains that exhibit disease produce two
different protein toxins: a strong cytotoxin and an
enterotoxin causing diarrhea; they are released by the
vegetative cells (Bielec & Modrzewska, 2007).
C. perfingens causes gas gangrene and is the cause of
severe food poisoning, associated with the release of
enterotoxin by vegetative cells. In the development of
gas gangrene other species of Clostridium are involved,
including C. novyi and C. septicum (Stackebrandt &
Hippe, 2001).
519
CONCLUSIONS
The Clostridium genera consist of many bacterial species with a wide range of physiological properties, thanks
to which they may be used in many branches of the industry. Despite the fact that a lot of Clostridium species
are pathogenic, some of them are used in medicine, in
cancer treatment, and in cosmetology to remove wrinkles. Moreover, enzymes isolated from these bacterial
cells are commonly used in modern biotechnology.
The important issue is to demonstrate that bacteria
from Clostridium spp. are not only pathogens and the enemy of humanity but they also have many beneficial and
important properties which make them usable among
other chemical, medical, and cosmetic applications.
Acknowledgement
The paper was prepared within the framework of project no. 01.01.02-00-074/09 co-funded by The European
Union from The European Regional Development Fund
within the framework of the Innovative Economy Operational Programme 2007–2013.
REFERENCES
Aldape MJ, Bryant AE, Stevens DL (2006) Clostridium sordellii infection:
Epidemiology, clinical findings, and current perspectives on diagnosis and treatment. Clin Infect Dis 43: 1436–1446.
Aureli P, Franciosa G, Fenicia L (2008) Botulism. International Encyclopedia of Public Health 329-337.
Bankar SB, Survase SA, Singhal RS, Granström T (2012) Continuous
two stage acetone–butanol–ethanol fermentation with integrated
solvent removal using Clostridium acetobutylicum B 5313. Bioresource
Technol 106: 110–116.
Barbé S, Van Mellaert L, Anné J (2006) The use of clostridial spores
for cancer treatment. J Appl Microbiol 101: 571–578.
Barber JM, Robb FT, Webster JR, Woods DR (1979) Bacteriocin production by Clostridium acetobutylicum in an industrial fermentation
process. Appl Environ Microb 37: 433–437.
Barnes MA, Ho AS, Malhotra PS, Koltai PJ, Messner A (2011) The
use of botulinum toxin for pediatric cricopharyngeal achalasia. Int J
Pediatr Otorhi 75: 1210–1214.
Beckers L, Hiligsmann S, Hamilton Ch, Masset J, Thonart P (2010)
Fermentative hydrogen production by Clostridium butyricum
CWBI1009 and Citrobacter freundii CWBI952 in pure and mixed cultures. Biotechnol Agron Soc Environ 14 (S2): 541–548.
Biebl H, Zeng AP, Menzel K, Deckwer WD (1998) Fermentation of
glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumonia.
Appl Microbiol Biot 50: 24-29.
Bielec D, Modrzewska R (2007) A history of botulin toxin diseases - clinical
aspects. Epidemiologic Rev 61: 505–512 (in Polish).
Bigalke H, Shoer LF (2000) “Clostridial neurotoxins” in Bacterial Protein Toxins In Handbook of Experimental Pharmacology, Aktories K,
Just I eds. 145: 407–443. Springer-Verlag, Berlin.
Blumer-Schuette SE, Kataeva I, Westpheling J, Adams MWW, Kelly
RM (2008) Extremely thermophilic microorganisms for biomass
conversion: status and prospects. Curr Opin Biotechnol 19: 210-217.
Brin MF (1997) Botulinum toxin: chemistry, pharmacology, toxicity,
and immunology. Muscle & Nerve 20: 156–168.
Buckel W (2005) Special “Clostridial enzymes and fermentation pathways” In Handbook on Clostridia, p 81. CRC Press LLC, Boca Raton.
Caya JG, Agni R, Miller JE (2004) Clostridium botulinum and the clinical laboratorian: a detailed review of botulism, including biological
warfare ramifications of botulinum toxin. Arch Pathol Lab Med 128:
653–662.
Celińska E (2010) Debottlenecking the 1,3-propanediol pathway by
metabolic engineering. Biotechnol Adv 28: 519–530.
Chen JS, Hiu SF (1986) Acetone-butanol-isopropanol production by
Clostridium beijerinckii (synonym Clostridium butylicum). Biotechnol Lett 8:
371–376.
Chen WM, Tseng ZJ, Lee KS, Hang JS (2005) Fermentative hydrogen
production with Clostridium butyricum CGS5 isolated from anaerobic
sewage sludge. Int J Hydrogen Energ 30: 1063–1070.
Cherington M (1998) Clinical spectrum of botulism. Muscle Nerve 21:
701–710.
Chin HL, Chen ZS, Chou CP (2003) Fed-batch operation using
Clostridium acetobutylicum suspension culture as biocatalyst for enhancing hydrogen production. Biotechnol Progr 19: 383–388.
520
D. Samul and others
Clarke DJ, Moyra RR, Morris JG (1975) Purification of two Clostridium
bacteriocins by procedures appropriate to hydrophobic proteins.
Antimicrob Agents Chemother 3: 256–264.
Colhado OCG, Boeing M, Ortega LB (2009) Toxina botulínica no
tratamento da dor botulinum toxin in pain treatment. Rev Bras Anestesiol 59: 366–381.
Dabrock B, Bahl H, Gottschalk G (1992) Parameters affecting solvent production by Clostridium pasteurianum. Appl Environ Microb 58:
1233–1239.
Demirbaş A (2001) Biomass resource facilities and biomass conversion processing for fuels and chemicals. Energ Convers Manage 42:
1357–1378.
Dressler D (2000) Botulinum toxin therapy, pp 64–67. Theme-Verlag,
Stuttgard.
Drożdżyńska A, Leja K, Czaczyk K (2011) Biotechnological production of 1,3-propanediol from crude glycerol. J Biotechnol, Comput Biol
Bionanotechnol 92: 92–100.
Dürre P (2001) From Pandora’s Box to Cornucopia: Clostridia — A Historical Perspective in Clostridia: biotechnology and medical applications, Bahl H,
Dürre P eds, pp 1–6; 20–22. Wiley-VCH Verlag GmbH, Germany.
Eklund FT, Poysky LM, Mseitif T, Strom MT (1988) Evidence for
plasmid-mediated toxin and bacteriocin production in Clostridium
botulinum Type G. Appl Environ Microb 54: 1405–1408.
Ezeji TC, Qureshi N, Blaschek HP (2003) Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping. World J Microb Biot 19: 595–603.
Friedmana A, Potulska A (2001) Quantitative assessment of parkinsonian sialorrhea and results of treatment with botulinum toxin. Parkinsonism Relat D 7: 329–332.
George HA, Johnson JL, Moore WEC, Holdeman LV, Chen JS (1983)
Acetone, isopropanol and butanol production by Clostridium beijerinckii (syn. C. butylicum) and Clostridium aurantibutyricum. Appl Environ
Microb 45: 1160–1163.
Glass GE, Hussain M, Fleming ANM, Powell BWEM (2009) Atrophy
of the intrinsic musculature of the hands associated with the use of
botulinum toxin-A injections for hyperhidrosis: a case report and
review of the literature. J Plast Reconstr Aesthet Surg 62: 274–276.
Gottumukkala LD, Parameswaran B, Valappil SK, Mathiyazhakan K,
Pandey A, Sukumaran RK (2013) Biobutanol production from rice
straw by a non acetone producing Clostridium sporogenes BE01. Bioresource Technol 145: 182–187.
Graham HK, Aoki KR, Autti-Rämö I, Boyd RN, Delgado MR, Gaebler-Spira DJ, Gormley Jr ME, Guyer BM, Heinen F, Holton AF,
Matthews D, Molenaers G, Motta F, Garcı Ruiz PJ, Wissel J (2000)
Recommendations for the use of botulinum toxin type A in the
management of cerebral palsy. Gait Posture 11: 67–79.
Hao J, Wei W, Jiesheng T, Jilun L, Dehua L (2008) Decrease of 3-hydroxypriopionaldehyde accumulation in 1,3-propanediol production
by over-expressing dhaT gebe in Klebsiella pneumonia TUAC01. J Ind
Microbiol Biot 35: 735–741.
Harris LM, Desai RP, Welker NE, Papoutsakis ET (1999) Characterization of recombinant strains of the Clostridium acetobutylicum butyrate
kinase inactivation mutant: Need for new phenomenological models
for solventogenesis and butanol inhibition. Biotechnol Bioeng 67: 1–11.
Heyndrickx, M, De Vos P, Vancanneyt M, De Ley J (1991) The fermentation of glycerol by Clostridium butyricum LMG 1212 t2 and 1213
t1, and C. pasteurianum LMG 3285. Appl Microbiol Biot 34: 637–642.
Igari S, Mori S, Takikawa Y (2000) Effects of molecular structure of
aliphatic diols and polyalkylene glycol as lubricants on the wear of
aluminum. Wear 244: 180–184.
Jagoda A, Renner G (1990) Infant botulism: Case report and clinical
update. AM J Emerg Med 8: 318–320.
Jankovic J, Hallet M (1994) Therapy with botulinum toxin. Marcel Dekker,
Inc, New York.
Jaspers GWC, Pijpe J, Jansma J (2011) The use of botulinum toxin
type A in cosmetic facial procedures. Int J Oral Max Surg 40: 127–
133.
Jiang L, Wang J, Liang S, Wang X, Cen P, Xu Z (2009) Butyric acid
fermentation in a fibrous bed bioreactor with immobilized Clostridium tyrobutyricum from cane molasses. Bioresource Technol 100: 3403–
3409.
Johns AT (1952) The mechanism of propionic acid formation by
Clostridium propionicum. J Gen Microbiol 6: 123–127.
Johnson EA, Bradshaw M (2001) Clostridium botulinum and its neurotoxin: a metabolic and cellular perspective. Toxicon 39: 1703–1722.
Johnson JL, Toth J, Santiwatanakul S, Chen JS (1997) Cultures of
Clostridium acetobutylicum from various collections comprise Clostridium acetobutylcium, Clostridium beijerinckii and two other distinct types
based on DNA-DNA reassociation. Int J Syst Bacteriol 47: 420–424.
Jones DL (1998) Organic acids in the rhizosphere — a critical review.
Plant Soil 205: 25–44.
Jones DT, van der Westhuizen A, Long S, Allcock ER, Reid SJ,
Woods DR (1982) Solvent production and morphological changes
in Clostridium acetobutylicum. Appl Environ Microb 43: 1434–1439.
Jones DT, Woods DR (1986) Acetone-butanol fermentation revisited.
Microbiol Rev 50: 481–524.
2013
Kasap M (2002) Nitrogen metabolism and solvent production in
Clostridium beijerinckii NRRL B593. Ph.D. dissertation. Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA.
Keis S, Shahneen R, Jones D (2001) Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii, and descriptions of
Clostridium saccharoperbutylacetonicum sp. nov. and Clostridium saccharobutylicum sp. nov. Int J Syst Evol Micr 51: 2095–2103.
Khanal SM, Chen WH, Sung S (2004) Biological hydrogen production:
effects of pH and intermediate products. Int J Hydrogen Energ 29:
1123–1131.
Kośmider A, Drożdżyńska A, Blaszka K, Leja K, Czaczyk K (2010)
Propionic acid production by Propionibacterium freudenreichii ssp. shermanii using industrial wastes: crude glycerol and whey lactose. Polish
J of Environ Stud 19: 1249–1253.
Kreydon OP, Geiges ML, Boni R, Burg G (2000) Botulinum toxin:
from poison to medicine. A historical review. Hautarzt 51: 733–737.
Ksibi I, Godard AL, Azouvi P, Denys P, Dziri C (2009) Botulinum
toxin and refractory non-neurogenic overactive detrusor. Ann Phys
Rehabil Med 52: 668–683.
Kubiak P, Leja K, Myszka K, Celińska E, Spychała M, SzymanowskaPowałowska D, Czaczyk K, Grajek W (2012) Physiological predisposition of various Clostridium species to synthetize 1,3-propanediol
from glycerol. Process Biochem 47: 1308–1319.
Lanigan GW (1959) Studies on the pectinolytic anaerobes Clostridium
flavum and Clostridium laniganii. J Bacteriol 77: 1–9.
Lehouritis P, Springer C, Tangney M (2013) Bacterial-directed enzyme
prodrug therapy. J Control Release 170: 120–131.
Leja K, Czaczyk K, Myszka K (2011) The use of microorganisms in
1,3-propanediol production. African J Microbiology Res 5: 4652–4658.
Levin BD, Islam R, Cicek N, Sparling R (2006) Hydrogen production
by Clostridium thermocellum 27405 from cellulosic biomass substrates.
Int J Hydrogen Energ 31: 1496–1503.
Liu X, Zhu Y, Yang ST (2006) Butyric acid and hydrogen production
by Clostridium tyrobutyricum ATCC 25755 and mutants. Enzyme Microb
Tech 38: 521–528.
Lo YCh, Lu WCh, Chen ChY, Chang JS (2010) Dark fermentative
hydrogen production from enzymatic hydrolysate of xylan and pretreated rice straw by Clostridium butyricum CGS5. Bioresource Technol
101: 5885–5891.
Luers F, Seyfried M, Daniel R, Gottschalk G (1997) Glycerol conversion by glycerol fermentation by Clostridium pasteurianum: cloning and
expression of the gene encoding 1,3-propanediol dehydrogenase.
FEMS Microbiol Lett 154: 337–345.
Mahajan ST, Brubaker L (2007) Botulinum toxin: from life-threatening
disease to novel medical therapy. Am J Obstet Gynecol 196: 7–15.
Majid OW (2010) Clinical use of botulinum toxins in oral and maxillofacial surgery. Int J Oral Max Surg 39: 197–207.
Masset J, Hiligsmann S, Hamiltoa Ch, Beckers L, Franck F, Thonart
P (2010) Effect of pH on glucose and starch fermentation in batch
and sequenced-batch mode with a recently isolated strain of hydrogen-producing Clostridium butyricum CWBI1009. Int J Hydrogen Energ
35: 3371–3378.
Mayr E (1969) Principles of systematic zoology. McGraw-Hill, New York.
McKendry P (2002 a) Energy production from biomass (part 1): overview of biomass. Bioresource Technol 83: 37-46.
McKendry P (2002 b) Energy production from biomass (part 2): conversion technologies. Bioresource Technol 83: 47–54.
Mellaert L, Barbe´S, Anne J (2006) Clostridium spores as anti-tumour
agents. Trends Microbiol 4: 190–196.
Minton NP, Mauchline ML, Lemmon MJ, Brehm JK, Fox M, Michael
NP, Giaccia A, Brown JM (1995) Chemotherapeutic tumour targeting using clostridial spores. Microbiol Rev 17: 357–364.
Moriishi K, Koura M, Abe N, Fujii N, Fujinaga Y, Inoue K, Ogumad
K (1996) Mosaic structures of neurotoxins produced from Clostridium botulinum strain NCTC 2916. FEMS Microbiol Lett 140: 151–158.
Mu Y, Teng H, Zhang DJ, Wang W, Xiu ZL (2006) Microbial production of 1,3-propanediol by Klebsiella pneumoniae using crude glycerol
from biodiesel preparations. Biotechnol Lett 28: 1755–1759.
Nakamura CE, Whited G (2003) Metabolic engineering for the microbial production of 1,3-propanediol. Curr Opin Biotech 14: 454-459.
Nakamura Y, Miyafuji H, Kawamoto H, Saka S (2011) Acetic acid fermentability with Clostridium thermoaceticum and Clostridium thermocellum
of standard compounds found in beech wood as produced in hotcompressed water. J Wood Sci 57: 331–337.
Nigel P, Minton J, Brown M, Lambin P, Anne J (2001) Clostridia in
Cancer Therapy In Clostridia: biotechnology and medical applications, Bahl
H, Dürre P eds, pp. 251–265 Wiley-VCH Verlag GmbH, Germany.
Oh SE, Zuo Y, Zhang H, Guiltinan MJ, Logan BE, Regan JM (2009)
Hydrogen production by Clostridium acetobutylicum ATCC 824 and
megaplasmid-deficient mutant M5 evaluated using a large headspace
volume technique. Int J Hydrogen Energ 34: 9347–9353.
Qureshi N, Schripsema J, Lienhardt J, Blaschek HP (2000) Continuous
solvent production by Clostridium beijerinckii BA101 immobilized by
adsorption onto brick. World J Microb Biot 16: 377–382.
Vol. 60 Roles of bacteria from the Clostridium genus
Patyar S, Joshi R, Prasad Byrav DS, Prakash A, Medhi B, Das BK
(2010) Bacteria in cancer therapy: a novel experimental strategy. J
Biomed Sci 17: 17–21.
Ranade VV (1989) Drug delivery systems 1. Site-specific drug delivery
using liposomes as carriers. J Cancer Pharmacol 29: 685–694.
Ren Z, Ward TE, Logan BE, Regan JM (2007) Characterization of the
cellulolytic and hydrogen-producing activities of six mesophilic Clostridium species. J Appl Microbiol 103: 2258–2266.
Rosetto O, Seveso M, Caccin P, Schiavo G, Montecucco C (2001) Tetanus and botulinum neurotoxins: turning bad guys into good by research. Toxicon 39: 27–41.
Schiavo G, Matteoli M, Montecucco C (2000) Neurotoxins affecting
neuroexocytosis. Physiol Rev 80: 717–766.
Shapiro RL, Hatheway C, Swerdlow DL (1998) Botulism in the United
States: A Clinical and Epidemiologic Review. Ann Intern Med 129:
221–228.
Simpson LL (2000) Identification of the characteristics that underlie
botulinum toxin potency: Implications for designing novel drugs.
Biochimie 82: 943–953.
Skonieczny MT, Yargeau V (2009) Biohydrogen production by Clostridium beijerinckii: effect of pH and substrate concentration. Int J Hydrogen Energ 34: 3288–3294.
Skrivanova E, Marounek M, Benda V, Brezina P (2006) Susceptibility
of Escherichia coli, Salmonella sp. and Clostridium perfringens to organic
acids and monolaurin. Vet Med-Czech 51: 81–88.
Sobel J (2005) Botulism. Clin Infect Dis 41: 1167–1173.
Song JH, Ventura JRS, Lee ChH, Jahng D (2011) Butyric Acid Production from Brown Algae Using Clostridium tyrobutyricum ATCC 25755.
Biotech Bioprocess Eng 16: 42-49.
St Jean AT, Zhang M, Forbes NS (2008) Bacterial therapies: completing the cancer treatment toolbox. Curr Opin Biotech 19: 511–517.
Sun Z, Liu S (2012) Production of n-butanol from concentrated
sugar maple hemicellulosic hydrolysate by Clostridia acetobutylicum
ATCC824. Biomass Bioenerg 39: 39–47.
521
Sutcliffe RP, Sandiford NA, Khawaja HT (2005) From frown lines to
fissures: Therapeutic uses for botulinum toxin. Int J Surg 3: 141–146.
Tong-Long Lin A (2007) Basic pharmacology of botulinum toxin in
the lower urinary tract. Tzu Chi Med J 19: 109–114.
Tracy BP, Jones SW, Fast AG, Indurthi DC, Papoutsakis ET (2012)
Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications. Curr Opin
Biotech 23: 364–381.
Tsui JKC (1996) Botulinum toxin as a therapeutic agent. Pharmacol Ther
72: 13–24.
Wei MQ, Mengesha A, Good D, Anne´J (2008) Bacterial targeted tumour therapy-dawn of a new era. Cancer Lett 259: 16–27.
Wheelera A, Smithb HS (2013) Botulinum toxins: Mechanisms of action, antinociception and clinical applications. Toxicology 306: 124–
–146.
Wilkens E, Ringel AK, Hortig D, Willke T, Vorlop KD (2012)
High-level production of 1,3-propanediol from crude glycerol by
Clostridium butyricum AKR102a. Appl Microbiol Biot 93: 1057–1063.
Willims EA, Coxhead JM, Mathers JC (2003) Anti-cancer effects of
butyrate: use of microarray technology to investigate mechanisms.
Proc Nutr Soc 62: 107–115.
Wu Z, Yang SH (2003) Extractive fermentation for butyric acid production from glucose by Clostridium tyrobutyricum. Biotech Bioeng 82:
93–102.
Zhu Y, Wu Z, Yang ST (2002) Butyric acid production from acid hydrolysate of corn fibre by Clostridium tyrobutyricum in a fibrous-bed
bioreactor. Process Biochem 38: 657–666.
Zigova J, Sturdik E, Dusan V, Schlosser S (1999) Butyric acid production by Clostridium butyricum with integrated extraction and pertraction. Process Biochem 34: 835–843.